The hypothesis underlying this proposal is that a primary mechanism that ensures maternal tolerance of the fetal allograft is the regulation of expression of Major Histocompatibility Complex (MHC) class I and class II antigens on the trophoblast cells that comprise the physical barrier between mother and fetus. The aim of this project is to characterize the expression and regulation of MHC class I genes in the trophoblast of equids during early pregnancy. We have chosen the horse as our experimental model because its trophoblast shows many morphological and endocrinological similarities to that of humans. Additionally, the form and tempo of placental development in equids allows a clarity of in vivo and in vitro investigations of immunological questions that cannot be matched in any other system. Advantages of the equine model include non- surgical recovery of conceptuses at any stage of development between days 6 and 36 after ovulation and ease of morphological identification and separation of trophoblast populations. Most importantly, simple methods have been devised for isolation of pure trophoblast populations for in vitro culture, allowing biochemical and molecular characterization of expressed molecules. Finally, our laboratories have between them the capacity to produce inter-species mule pregnancies and fully xenogeneic donkey-in-horse pregnancies by embryo transfer. These provide unique models of pregnancy and pregnancy failure for the study of immunological factors that govern fetal-maternal interactions at implantation. We propose to investigate different aspects of MHC gene expression and regulation using 3 distinct populations of equine trophoblast: i) in MHC class I (+) cytotrophoblast (chorionic girdle), we would delineate the number and type of class I genes expressed, and- the temporal pattern of expression and coordinate regulation of MHC and beta-2 microglobulin genes; ii) in MHC class I(-) cytotrophoblast (allantochorion), we would determine the factors which bring about the MHC class I negative state and those that can cause up regulation in this population, and iii) in MHC class I(-) binucleate trophoblast, we would investigate whether the apparent irreversible down regulation of MHC genes is brought about by mechanisms other than those operating in (ii). A large number of horse-specific reagents are already in hand. These include monoclonal antibodies and alloantisera to MHC class I antigens, cDNA and genomic libraries, full length MHC class I cDNA and genomic clones, and stable, fully characterized MHC class I transfectants. In addition, horses of defined MHC haplotypes, including homozygotes, are available for experimental matings. It is probable that the mechanisms which regulate expression of MHC genes in trophoblast cells are conserved between species. Our proposed studies of equine pregnancy are, therefore, likely to shed light on mechanisms that operate in human pregnancy. They may also have relevance to MHC gene regulation in tumor growth and transplantation biology.